Amyotrophic lateral sclerosis (ALS) is a progressive and/or tardive neurodegenerative disease, by which upper and lower motor neurons are selectively damaged. Amyotrophic lateral sclerosis is an extremely serious disease, which has symptoms such as convulsion, muscle paralysis or muscle atrophy, brings on respiratory failure mainly caused by respiratory muscle paralysis, and results in death in a few years after the onset thereof. The number of patients suffering from amyotrophic lateral sclerosis in Japan is approximately 9000, and there are only several hundreds of thousands of ALS patients over the world. Since the number of ALS patients is fewer than those of patients with other neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease, the development of therapeutic agents for ALS is behind. The currently approved therapeutic agents are only a glutamate nerve ending release inhibitor (Riluzole) and an active oxygen scavenger (Edaravone), which have the effect of temporally delaying progression (Non Patent Literatures 1, 2 and 3). However, the action mechanism of such neuron-protecting effect on ALS has not been elucidated. In addition, since the pathological molecular mechanism of developing ALS has not yet been known, a fundamental treatment method, which is based on a clear molecular basis, has not existed.
A majority of ALS diseases are sporadic ALS (SALS), and familial ALS (FALS) accounts for 10% of the whole ALS diseases. In 1993, Cu, Zn Superoxide dismutase (SOD1) has been identified for the first time as a causative gene of FALS (Non Patent Literature 4). More than 100 types of ALS-related SOD1 mutations have been reported so far (ALS Online Database: ALSoD). SOD1 is a metalloprotein having a disulfide bond in a molecule thereof, in which one Zn ion and one Cu ion are coordinated. It has been known that SOD1 generally forms a homodimer and acts as an antioxidant enzyme disproportionating active oxygen (O2) to hydrogen peroxide (H2O2) via the oxidation-reduction of Cu ions in an active center. On the other hand, a large number of studies have been conducted so far, regarding the correlation of SOD1 with the onset of ALS, and as a result, it has been revealed that there is no correlation between the antioxidative activity of SOD1 and the seriousness of the pathological condition of ALS (Non Patent Literature 5). At present, it is considered that not only the loss or acceleration of SOD activity, but also newly acquired cytotoxicity that is exhibited by SOD1 as a result of mutation has a certain importance on motor neuron death and the subsequent onset of ALS (Non Patent Literature 6). It has been suggested that the acquired cytotoxicity, such as neural excitotoxicity, endoplasmic reticulum stress, proteasome inhibition, oxidative stress, mitochondrial dysfunction or axonal transport abnormality, is associated with motor neuron death, but the detailed mechanism of the onset of ALS still remains unknown (Non Patent Literature 7).
Endoplasmic reticulum is an organelle that plays roles, such as formation of the conformation of a newly synthesized secretory protein or membrane protein, and the quality control of these proteins. Among the proteins existing in the endoplasmic reticulum lumen, proteins that do not have a correct conformation, namely, unfolded proteins are present at a certain ratio. In addition, such unfolded proteins are formed even by various physiological and/or pathological stresses, such as nutrient starvation, hypoxia or gene mutation, and the thus formed unfolded proteins are accumulated in the endoplasmic reticulum lumen, so that they cause endoplasmic reticulum stress.
It has been known that, at this time, the mechanism of controlling endoplasmic reticulum quality is functioned in a cell by endoplasmic reticulum stress response (unfold protein response: UPR), in order to maintain normal function in the cell (Non Patent Literature 8). According to the UPR, signals are transmitted via activation of three types of endoplasmic reticulum transmembrane receptors, namely, Pancreatic ER kinase (PKR)-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1), so that (i) suppression of protein synthesis, (ii) induction of the expression of endoplasmic reticulum chaperones and molecules involved in endoplasmic reticulum-associated degradation (ER-associated degradation: ERAD), (iii) degradation of unfolded proteins by ERAD, etc. are carried out, and thereby, the recovery of the functions of the endoplasmic reticulum is achieved. On the other hand, when unfolded proteins are excessively accumulated in the endoplasmic reticulum lumen and the homeostasis of the endoplasmic reticulum cannot be maintained by continuous or excessive stress or the dysfunction of UPR, apoptosis is induced. It has been suggested that this endoplasmic reticulum stress-induced cell death should be involved, as a molecular mechanism of disease caused by accumulation of structurally abnormal proteins, in various pathologic conditions including diabetes or neurodegenerative disease (Non Patent Literature 9). However, the molecular mechanism of inducing endoplasmic reticulum stress in ALS has not yet been elucidated.
The present inventors had reported that, as a mechanism of the onset of FALS, the function of ERAD is inhibited by mutant SOD1 and thereby, motor neuron death caused by endoplasmic reticulum stress is induced (Non Patent Literature 10). In general, unfolded proteins existing in the endoplasmic reticulum lumen, which could not have a correct conformation even by the function of endoplasmic reticulum chaperones, are decomposed by ERAD. Specifically, such unfolded proteins are recognized by endoplasmic reticulum chaperones including BiP as a typical example, and are then reversely transferred from the endoplasmic reticulum lumen to the cytoplasmic side via an ERAD complex. Thereafter, the unfolded proteins are decomposed by an ubiquitin-proteasome system existing on the cytoplasmic side, so that the accumulation of the unfolded proteins in the endoplasmic reticulum lumen is reduced, and the homeostasis of the endoplasmic reticulum is maintained (Non Patent Literatures 11 and 12). On the other hand, in cells in which mutant SOD1 is expressed, it has been found that such mutant SOD1 specifically binds to the C-terminal 12 amino acids (hereinafter abbreviated as “CT4”) of Derlin-1 that is an important configuration factor of the ERAD complex, and it inhibits the flow of ERAD, so as to suppress decomposition of the ERAD substrate. Due to such inhibition of the function of ERAD, unfolded proteins are accumulated in the endoplasmic reticulum lumen, and the thus induced endoplasmic reticulum stress causes the death of motor neurons via apoptosis (Non Patent Literature 10).
Derlin-1 is an endoplasmic reticulum transmembrane protein that has been identified as a molecule playing an important role in the retrotransloction of the ERAD substrate (Non Patent Literatures 13 and 14). It has been demonstrated that Derlin-1 homologs in mammals include Derlin-2 and Derlin-3, and that these Derlin family molecules form a homo or hetero complex, and interact with ERAD-related molecules such as HRD1, SEL1L, Herp, VIMP, or p97 (Non Patent Literatures 15, 16 and 17). Details of the function of Derlin-1 in ERAD have not yet been revealed, but it has been found that ALS-related mutant SOD1 does not bind to Derlin-2 or Derlin-3, or ERAD-related molecules such as VIMP or p97, but that ALS-related mutant SOD1 specifically binds to only the C-terminus of Derlin-1. Moreover, suppression of the expression of Derlin-1 by knocking down did not provoke endoplasmic reticulum stress, but such suppression of Derlin-1 expression inhibited induction of endoplasmic reticulum stress by mutant SOD1 (Non Patent Literature 10). From these results, it is considered that endoplasmic reticulum stress caused by mutant SOD1 is not induced via the loss of the function of Derlin-1, but the endoplasmic reticulum stress is induced, when Derlin-1 binds to mutant SOD1 so that the Derlin-1 exhibits acquired dysfunction and normal ERAD is thereby inhibited.
The present inventors have produced expression vectors for 130 types of known ALS-related mutant SOD1s, and have examined the binding between all of the mutant SOD1s and Derlin-1. As a result, it has been revealed that all of the mutant SOD1s except for mutants having very low correlation with the pathologic conditions (i.e., 122 types of mutant SOD1s) bind to Derlin-1 CT4 (Non Patent Literature 18). Since mutation covers a broad range on the primary sequence of SOD1, it has been considered that each mutant SOD1 does not individually acquire a surface binding to Derlin-1 as a result of the mutation, but that a region binding to Derlin-1 is present in the sequence of wild-type SOD1. As such, the region of SOD1 that interacts with Derlin-1 has been examined. As a result, it has been revealed that a region binding to Derlin-1 (Derlin-1 binding region: DBR) consisting of 14 amino acids is present in wild-type SOD1, as expected (Non Patent Literature 18). Moreover, a monoclonal antibody produced against this DBR region did not recognize wild-type SOD1, but specifically recognized all mutant SOD is that bind to Derlin-1 (except for mutations of antibody epitope portions) in an immunoprecipitation experiment (Non Patent Literature 18 and Patent Literature 1). From these results, it has been suggested that ALS-related mutant SOD1s should be likely to have a common feature that DBR, which is hiding inside for the conformational reason in the case of wild-type SOD1, is exposed outside due to a structural change caused by mutation. Furthermore, SOD1 in a B cell derived from a human ALS patient having an SOD1 gene mutation has also been recognized by a mutant SOD1-specific antibody (Non Patent Literature 18 and Patent Literature 1), and it has also been confirmed that endogenous SOD1 and Derlin-1 actually bind to each other. Accordingly, it has been considered that the binding of SOD1 to Derlin-1 via DBR exposure is also associated with the pathologic conditions of human ALS. In addition, it has been revealed that, in a mouse spinal cord-derived primary motor neuron culture system, mutant SOD1-induced motor neuron death is suppressed by allowing a peptide that inhibits the binding between SOD1 and Derlin-1 (Derlin-1 (CT4) peptide: Derlin-1 CT4 region-derived peptide) to express in the cells (Non Patent Literature 10).
As mentioned above, it has been suggested that inhibition of the binding between ALS-related mutant SOD1 and Derlin-1 should lead to the treatment of ALS caused by SOD1 mutation. However, a therapeutic agent and a treatment method, which are effective for ALS, have not yet been discovered so far.